Method for modifying lignin structure using monolignol ferulate conjugates

ABSTRACT

Described is an isolated lignified plant cell wall including lignin, wherein the lignin includes a ferulate residue incorporated therein, such as from coniferyl ferulate and/or sinapyl ferulate. Also described is a method to make the isolated lignified plant cell wall, and the lignin produced by the method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to U.S.Provisional Patent Application 61/213,706 filed Jul. 6, 2009, theentirety of which is incorporated herein by reference.

REFERENCES

All of the documents cited herein are incorporated herein by reference.

INTRODUCTION

Lignin is a highly complex, heterogeneous polymer found in all vascularplants. It rigidifies plants and plays a crucial role in watertransport. Lignin is notable for its complex structure. It is comprisedpredominately from three monomers, p-coumaryl alcohol 1, coniferylalcohol 2, and sinapyl alcohol 3, and a host of other structurallyrelated monomers. See FIG. 1. Other typical monomers found in naturallignins include 5-hydroxyconiferyl alcohol, hydroxycinnamaldehydes,hydroxybenzaldehydes, arylglycerols, tyramine hydroxycinnamates,hydroxycinnamic acids, hydroxycinnamate esters, dihydro-hydroxycinnamylalcohols, arylpropane-1,3-diols, and various acylatedmonolignols-hydroxycinnamyl acetates,hydroxycinnamyl-p-hydroxybenzoates, and hydroxycinnamyl-p-coumarates.Hydroxycinnamaldehydes and their corresponding hydroxybenzaldehydes arefound in all lignins. Hydroxycinnamyl acetates are found in mosthardwoods and are present in high levels in kenaf and palms.Hydroxycinnamyl p-hydroxybenzoates are found in willows, palms, poplars,and aspens. Hydroxycinnamyl p-coumarates are found in all grasses. Thesemonomers are polymerized into polymeric lignin by combinatorial radicalcoupling reactions. The lignification of cell walls is also notablebecause it is likely the single most important factor that limits foragedigestibility in ruminants and the “saccharification” of plantpolysaccharides to simple sugars for use in biofuel or biochemicalapplications. Practically speaking, lignin is indigestible in thedigestive tract of ruminants. The interfering presence of indigestiblelignin limits the ability of ruminants to utilize otherwise digestiblecarbohydrates present in the forage they eat. Lignin also limits enzymeaccess to cell wall polysaccharides, inhibiting the release ofmonosaccharides for conversion to other products including biofuels.Thus, there remains a long-felt and unmet need to alter lignins in sucha way that improves the digestibility/fermentability of the cell wallpolysaccharides.

Over the past decade it has become apparent that the metabolicmalleability of lignification, the process of polymerization of phenolicmonomers to produce lignin polymers, provides enormous potential forengineering the troublesome polymer to be more amenable to processing.Massive compositional changes can be realized by perturbing single genesin the monolignol pathway, particularly the hydroxylases (Ralph, et al.Phytochem. Revs. (2004), 3(1), 29-60; Boerjan, et al. Annu. Rev. PlantBiol. (2003), 54, 519-549; Marita, et al. Proc. Natl. Acad. Sci. (1999),96(22), 12328-12332; Franke, et al. Plant J. (2002), 30(1), 47-59). Thechemical nature of lignification, involving combinatorial radicalcoupling of monomers (primarily with the growing polymer) without directenzymatic control, allows compatible phenolic compounds present in thecell wall (CW) during lignification to be incorporated into the “lignin”polymer. Novel (non-monolignol) monomers available to the plant,discovered in lignins from studies on the down-regulation of genes inthe monolignol pathway, include products of incomplete monolignolbiosynthesis such as 5-hydroxyconiferyl alcohol (COMT-deficiency), andconiferaldehyde and sinapaldehyde (CAD-deficiency) (Ralph et al.Phytochem. (2001), 57(6), 993-1003). These compounds couple integrally(via a radical route) into the lignin polymer. The list of othercompounds found integrated into lignins in normal and/or transgenicplants is growing (Boerjan, et al. Annu. Rev. Plant Biol. (2003), 54,519-549).

Observations to date have allowed the present inventors to detail theideal properties of monolignol substitutes (Ralph, J., What makes a goodmonolignol substitute? In The Science and Lore of the Plant Cell WallBiosynthesis, Structure and Function, Hayashi, T., Ed. UniversalPublishers, BrownWalker Press: Boca Raton, Fla., (2006); pp 285-293).When such compounds are introduced into lignins, even at significantlevels, the plants often show no obvious growth/development phenotype.Monomers that have accessible conjugation into the sidechain allowingfor so-called “endwise” β-O-4-coupling seem to fare the best. Examplesare: 5-hydroxyconiferyl alcohol, the hydroxycinnamaldehydes,hydroxycinnamate esters, and acylated hydroxycinnamyl alcohols. Due toincompatibilities in radical coupling reactions, p-hydroxyphenylmoieties fare less well than guaiacyl or syringyl moieties, at leastwhen incorporating into guaiacyl-syringyl lignins, but other phenolicshave not been well studied.

Replacing the entire monomer component of lignification with a novelmonomer is unlikely to be an effective strategy that is “acceptable” tothe growing plant. Introducing strategic monomers into the normalmonolignol pool is, however, a viable proposition as shown by theExamples described herein. Incorporation of novel monomer residues intolignin as described herein has produced plants with no pleiotropiceffects or obvious growth phenotypes. Incorporation of up to 60% novelmonomer residues into lignin has been accomplished. A range ofalternative monomers are shown herein to be consistent with maintainingthe plant's structural and functional integrity. Thus, the crux of thepresent invention is a method of manufacturing modified lignin usingmonomer-conjugate substitution (as well as the resulting modified ligninpolymer itself). The resulting modified lignin drastically easesprocessing of the cell wall to yield value-added products, such asanimal feeds and forages, pulps for papermaking, fermentable substratesfor biofuel and chemical production, and the like.

SUMMARY OF THE INVENTION

Disclosed and claimed herein is a method of manufacturing modifiedlignin, the lignin so produced, and isolated cell walls incorporatingthe modified lignin.

Specifically, a first version of the invention is directed to anisolated lignified plant cell wall comprising lignin, wherein the ligninincludes a ferulate residue incorporated therein. In a preferredversion, the ferulate residue is introduced via monolignol-ferulateconjugates selected from the group consisting of coniferyl ferulate,sinapyl ferulate, and other structurally related conjugates.

The plant cell wall from which the lignin is isolated may be derivedfrom any plant source, without limitation. That is, the plant cellsthemselves may be derived from any species of the Plantae kingdom thatis now known to make lignin naturally, is discovered in the future tomake lignin naturally, or does not make lignin naturally, but has beengenetically modified to make lignin, without limitation. The plant cellsmay also be derived from plants that have been genetically modified forother purposes. This includes vascular plants of all description,monocots and dicots, hardwood and softwood trees, shrubs, grasses,grains, fruits, vegetables, etc. Preferred sources are grasses such asmaize, Miscanthus, sorghum, and switchgrass, and trees, such as trees ofthe family Myrtaceae, or hybrids thereof, including trees of the generaEucalyptus, Corymbia, and Angophora, as well as trees of the familySalicaceae, including trees of the genera Populus (e.g., poplar, aspen,and cottonwood trees, etc.), and Salix (e.g., willow trees), or hybridsof any of the foregoing.

The preferred ferulate monomer conjugates are coniferyl ferulate andsinapyl ferulate.

Another version of the invention is directed to a method ofmanufacturing modified lignin. Here, the method comprises conducting alignin-producing polymerization reaction in the presence of at least onepolymerizable conjugate (preferably coniferyl ferulate and/or sinapylferulate) comprising a hydroxycinnamic or hydroxybenzoic acid (or ahydroxyl, alkyl, alkyloxy, alkanoyl, or alkanoyloxy derivative thereof)esterified to a hydroxycinnamyl or benzyl alcohol (or a hydroxyl, alkyl,alkyloxy, alkanoyl, alkanoyloxy, derivative thereof) wherein at leastone of the polymerizable conjugates is incorporated into the resultinglignin. Again, the preferred conjugates are coniferyl ferulate andsinapyl ferulate. It is preferred that from about 10% by wt to about 60%by wt of the polymerizable conjugates are reacted in the polymerizationreaction. The polymerization reaction is conducted in vitro or in vivo.

In one specific version of the invention, the polymerization reactioncomprises isolating a cell wall from a plant cell suspension andlignifying the cell wall in the presence of the polymerizable conjugate.

Included within the scope of the invention disclosed and claimed hereinis the modified lignin produced by the process.

As noted above, it is generally preferred that from about 10% by wt toabout 60% by wt of the polymerizable conjugates are reacted in thepolymerization reaction, although ranges above and below the statedrange are explicitly within the scope of the method (e.g., from 0.1 wt %to 100 wt %).

Also disclosed herein are isolated lignified cell walls containing acompound as recited above, wherein the compound is incorporated into thelignin of the cell wall. Likewise disclosed herein are isolated plantcells containing a compound as recited above, wherein the compound isincorporated into lignin in cell walls of the isolated plant cells.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a fully saturated, straight, branched chain, orcyclic hydrocarbon radical, or combination thereof, and can include di-and multi-valent radicals, having the number of carbon atoms designated(e.g., C₁-C₁₀ means from one to ten carbon atoms, inclusive). Examplesof alkyl groups include, without limitation, methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)ethyl, cyclopropylmethyl, and homologs, and isomers thereof,for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Theterm “alkyl,” unless otherwise noted, also includes those derivatives ofalkyl defined in more detail below as “heteroalkyl” and “cycloalkyl.”

The term “alkenyl” means an alkyl group as defined above containing oneor more double bonds. Examples of alkenyl groups include vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), etc., and higher homologs and isomers.

The term “alkynyl” means an alkyl or alkenyl group as defined abovecontaining one or more triple bonds. Examples of alkynyl groups includeethynyl, 1- and 3-propynyl, 3-butynyl, and the like, including higherhomologs and isomers.

The terms “alkylene,” “alkenylene,” and “alkynylene,” alone or as partof another substituent means a divalent radical derived from an alkyl,alkenyl, or alkynyl group, respectively, as exemplified by—CH₂CH₂CH₂CH₂—.

The term “hydroxy” is used herein to refer to the group —OH.

The term “alkoxy” is used herein to refer to the —OR group, where R isalkyl, alkenyl, or alkynyl, or a substituted analog thereof. Suitablealkoxy radicals include, for example, methoxy, ethoxy, t-butoxy, etc.The term “alkoxyalkyl” refers to ether substituents, monovalent ordivalent, e.g. —CH₂—O—CH₃ and —CH₂—O—CH₂—.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, 5, 6,from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice-versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods of the present invention can comprise, consist of, orconsist essentially of the essential elements and limitations of themethod described herein, as well as any additional or optionalingredients, components, or limitations described herein or otherwiseuseful in synthetic organic chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Structures of p-coumaryl alcohol 1, coniferyl alcohol 2, sinapylalcohol 3, ferulate 4, sinapyl p-coumarate 5, coniferyl ferulate 6, andsinapyl ferulate 7. Arrows indicate sites normally involved in radicalcoupling reactions during lignification.

FIG. 2: Copolymerization of coniferyl ferulate with monolignols to formlignin with ester linkages in the backbone of the polymer. Such linkagesfacilitate lignin depolymerization during alkaline or acidicpretreatment of biomass for saccharification or paper production.

FIG. 3: UV spectra of cell wall and dehydrogenation polymer (DHP)lignins prepared with coniferyl alcohol (CA) and coniferyl ferulate(CA-FA) and fully solubilized by 0.5 M aqueous NaOH at 160° C.

FIG. 4: Table 1. Concentrations (mg/g) of lignin, alkali-labileferulates, and total ferulates in cell walls.

FIGS. 5A and 5B: Delignification (FIG. 5A) of alkali-soluble lignin andfiber yield (FIG. 5B) by aqueous 0.5 M NaOH at 30° C. (22 h), 100° C.(2.5 h), or 160° C. (1.5 h, with anthraquinone) from artificiallylignified cell walls of maize prepared with 0 to 60% coniferyl ferulate.Bars indicate±SEM.

FIG. 6: Table 2. Concentrations (mg/g) of alkali-soluble lignin,alkali-soluble carbohydrate, and alkali-insoluble residue.

FIG. 7: Molecular weight distribution of alkali-soluble lignins releasedfrom maize cell walls by aqueous 0.5 M NaOH at 30, 100, or 160° C. Dataare averaged over cell walls artificially lignified with 0-60% coniferylferulate. Means within a molecular weight group with unlike lettersdiffer (P<0.05).

FIG. 8: Molecular weight distribution of alkali-soluble lignins fromartificially lignified cell walls of maize prepared with 0-60% coniferylferulate. Data are averaged over 0.5 M NaOH treatments at 30, 100, and160° C. Means within a molecular weight group with unlike letters differ(P<0.05).

FIG. 9: Impact of forming lignins with 0-60% coniferyl ferulate on thelignin content of non-treated (NT) cell walls and of alkali-insolubleresidues recovered following hydrolysis at 30, 100, or 160° C. Barsindicate±SEM.

FIG. 10: Table 3. Carbohydrate (mg/g) released from maize cell walls andalkali-insoluble residues (AIR) by enzymatic hydrolysis, andcarbohydrate (mg/g) released enzymatically from AIR plus alkali-solublecarbohydrate (AIR+ASC).

FIG. 11A: Relationship between lignin content and carbohydrate releasedafter a two-hour (2 h) enzymatic hydrolysis of cell walls (lignifiedwith 0-60% coniferyl ferulate or nonlignified) and theiralkali-insoluble residues prepared with 0.5 M NaOH at 30° C. Barsindicate±SEM.

FIG. 11B: Relationship between lignin content and carbohydrate releasedafter a forty-eight-hour (48 h) enzymatic hydrolysis of cell walls(lignified with 0-60% coniferyl ferulate or nonlignified) and theiralkali-insoluble residues prepared with 0.5 M NaOH at 30° C. Barsindicate±SEM.

DETAILED DESCRIPTION

Novel monomers that appear to be well suited for lignification can befound throughout the plant kingdom. Several groups of compounds, asdescribed below, are suitable for producing modified lignins. As ageneral proposition, the most suitable monomer for producing modifiedlignins fall into five (5) classes: (1) bifunctional monomers or monomerconjugates linked via cleavable ester or amide bonds; (2) monomers thatproduce novel cleavable functionalities in the lignin polymer; (3)hydrophilic monomers; (4) monomers that minimize lignin-polysaccharidecross-linking; and (5) monomers that produce simpler lignins. Each ofthese classes of monomers will be described below. In each instance,suitable monomers are polymerized into a modified lignin. The modifiedlignins are then assessed to see whether and how the modified ligninsimpact biomass processing in biomimetic cell wall systems.

Many of the experimental procedures referenced herein are described onlybriefly. Full-text references describing the known procedures can befound at: http://www.dfrc.ars.usda.gov/DFRCWebPDFs/pdfindex.html.

Classes of Monomers to Consider:

Without regard to plant biochemistry, it is straightforward to proposesuitable monomers from simple chemical principles. However, the onlymonomers that should be considered are those that plants canbiosynthesize; i.e., those compounds for which biosynthetic pathwaysexist. The following five classes of monomers are preferred for use inthe present invention:

Bifunctional Monomers or Monomer Conjugates Linked Via Cleavable Esteror Amide (and/or Hydrophilic) Functionality:

Monomers or conjugates with compatible phenolic groups at both ends areintroduced into the lignin polymer. Using these types of bifunctionalmonomers allows lignification to proceed in both directions toincorporate the monomer. If coupling at the sidechain β-position ispossible, such units can form important branch-points in the polymer.More importantly, if these units are linked by linkages that are readilycleaved during anticipated processing, they introduce groups into thepolymer allowing it to be readily “unzipped”—i.e., depolymerized. As aresult, delignification can therefore be achieved under less stringentconditions, releasing the polysaccharides with lower energy requirementsand higher yields.

Diferuloylated Compounds:

Ferulates incorporate integrally into lignins by the same types ofradical coupling reactions that typify lignification (Ralph, J. et al.Phytochem. Revs. (2004), 3(1), 79-96). Due to the combinatorial natureof such coupling reactions, the array of products is quite complex, butreadily determined by NMR experiments. Having two ferulates in amonolignol-substitute allows lignification to proceed from both. Theadvantage is that, even after both ferulates have been integrallyincorporated into the complex polymer, simply cleaving the esters thencleaves the polymer into fragments of lower molecular weight allowingtheir removal from the polysaccharides in the wall.

Bi-phenolic Conjugates:

Compounds that have two lignin-compatible moieties directly connectedvia cleavable functionality can also be incorporated into a modifiedlignin. For example, 3-methoxytyramine ferulate, unlike its tyramineferulate analog, will polymerize integrally into lignins at both ends.The benzylic-CH₂ means that the monomer unit may undergo some furtherconversion, via a quinone methide, to the benzylic-OH analog first, butboth this product and the parent will lignify. This benzylic conversionis analogous to the incorporation of dihydroconiferyl alcohol, and thederived guaiacylpropane-1,3-diol, into pine lignins, particularly in aCAD-deficient pine that produces enhanced levels of dihydroconiferylalcohol monomer (Ralph, J. et al. Org. Lett. (1999), 1(2), 323-326;Ralph, J. et al. Science (1997), 277, 235-239). However, unlikedihydroconiferyl alcohol monomer, which can only appear as an end-unitin lignins, the difunctional nature of methoxytyramine ferulate meansthat it can become fully integrated into the polymer chain. The monomerhas built-in bi-functionality. Finally, there are various ways ofcleaving amides under mild conditions. Utilization of thismonomer-substitute therefore is a way of introducing bond into theresultant lignin that can be readily cleaved during processing.

In this class are also various catechol derivatives, such as clovamide,caffeoyl 3-hydroxytyrosine, and rosmarinic acid. Catechol structureshave not previously, however, been validated in lignins. Despiterepeated attempts to find evidence for related caffeyl alcoholincorporation, even in CCOMT-deficient plants, there is currently noevidence for the natural incorporation of such non-methoxylatedcatechols into lignins. However, the related 3-methoxy-4,5-dihydroxycompounds (such as those produced in COMT-deficient plants that utilize5-hydroxyconiferyl alcohol as a monomer) integrate readily. Thus, thesecompounds are suitable monomers for lignification. These monomers mayprove more successful in p-hydroxyphenyl-rich lignification, in whichcase the target plants will also need to be C3H-deficient.

Methods for Testing Novel Lignification and the Resulting BiomassProcessing Improvements:

Delineate Monomer Compatibility:

Determining the compatibility of the chosen monomers with lignificationvia in vitro model coupling reactions is essential to determine as anyselected monomer that does not couple integrally into lignins isunlikely to be valuable. Coupling and cross-coupling propensities arebest tested empirically as there do not appear to be any systematicrules the predict whether a monomer will couple and cross-coupleappropriately. Such methods have been used to define how ferulatescouple into lignins, for example (Ralph, J. et al. J. Chem. Soc., PerkinTrans. 1 (1992), (21), 2961-2969). The models and model polymers willalso provide the NMR database required to identify how monomersincorporate into the more complex cell wall models and in transformedplants.

Biomimetically Lignify the Selected Monomers into Cell Walls:

Selected monomers, at varying levels relative to the normal monolignols,are incorporated into cell walls. Strategically ¹³C-labeled monomers areused as appropriate.

Delineate the Resultant Cell Wall Lignin Structure:

Structural characterization of the cell walls reveal whether themonomers integrate into wall lignins and also provide materials forconversion testing. Structures are examined by degradative methods and,most importantly, via the whole-cell-wall dissolution and NMR procedures(where the strategic labeling helps reveal the bonding patterns) (Lu, etal. Plant J. (2003), 35(4), 535-544).

Test Biomass Processing Impacts:

Monomers are selected for their potential to improve biomass processingefficiency. The cell walls created are tested under a variety of biomassconversion methods to delineate how much improvement might be expectedin planta from utilization of the monomer substitutes are variouslevels.

These steps are described more fully below.

Delineating Monomer Compatibility:

Synthetic in vitro coupling reactions, although they do not providematerials suitable for testing the effects of lignin modification, playa valuable role in the initial selection of potential monomers. Thereasoning is simple. All the coupling reactions evidenced in lignins invivo are also produced, admittedly at different relative levels, invitro. If coupling and cross-coupling compatibility is not observed insynthetic coupling reactions, failure in planta is almost certainlyassured because the in vivo reaction is also purely chemical. Becausethe monomer-substitutes are envisioned to incorporate into lignificationwith the normal monolignols, it is important that they be compatiblewith coupling and, more importantly, cross-coupling reactions with thegrowing polymer derived in part from those monolignols.

It is for these reasons that some suggestions, seemingly logical onpaper, will not function. For example, it has already been establishedthat non-methoxylated phenolic entities such a tyramine and p-coumaratedo not become integrated into the polymer by coupling reactions. Theyare found in lignin polymers, but only as “appendages” or end-units. Forexample, p-coumarates are exclusively found as acylating groups inlignin sidechain γ-positions. They are free-phenolic (non-etherified),meaning that they do not undergo radical coupling reactions. On theirown, in vitro, p-coumarates will couple, but what happens duringlignification in the presence of normal monolignols and ligninguaiacyl/syringyl units is that radical transfer from these less-stableradicals occurs before they will enter into radical coupling (Ralph, J.et al. Phytochem. Revs. (2004), 3(1), 79-96). Thus, lignifying withconiferyl- or sinapyl p-coumarate is known not to work. The coniferyland sinapyl alcohol moieties incorporate as usual, but the p-coumarateend, despite being phenolic and potentially capable of radical coupling,will not incorporate—the units remain as free-phenolic pendant units. Asa result, cleaving the esters will release the p-coumarate but will notcause any depolymerization of the polymer. Similarly, the idea of usingtyramine ferulate will not work either; tyramine units (alsonon-methoxylated phenolics) do not enter into coupling reactions undernormal in planta conditions (Ralph, J. et al. Proc. Natl. Acad. Sci.(1998), 95(22), 12803-12808). However, if lignins are derived fromhigher levels of the non-methoxylated monolignol, p-coumaryl alcohol,p-coumarates and tyramines will cross-couple into thosep-hydroxyphenyl-rich polymers. Thus exploring the chemical compatibilityof monomers first will delineate whether it is worth introducing thosemonomers into C3H-deficient plants, for example—plants in which theconiferyl and sinapyl alcohol levels are depleted at the expense of thepotentially compatible p-coumaryl alcohol.

Although tyramine ferulate was noted as not being a candidate forintroducing cleavable bonds into lignins, an analog can be found incertain plants. 3-Methoxytyramine ferulate is a bifunctional molecule inwhich both moieties are entirely compatible with lignification. Ittherefore incorporates fully, from both ends, into lignin. The cleavableamide functionality then introduced into the backbone of the polymer isexactly the kind of zipper unit that will allow such a polymer to bemore readily depolymerized.

Biomimetic Lignification into Suspension-Cultured Cell Walls:

Once the monomers have been obtained/synthesized, they are then testedfor their lignifying ability. As a general rule, it is not preferred tomake synthetic lignins by simple in vitro polymerization of thesemonomers (with or without the traditional monomers) because the in vitromaterials give little insight into the behavior of the cell wall duringbiomass processing. It is much preferred to produce cell walls lignifiedwith the novel monomers (either in the presence of, or the absence ofthe normally present monolignols). A suspension-cultured corn system forproducing cell walls amenable to controlled lignification by exogenouslysupplying the lignin monomers has been described. See Grabber, J. H. etal. J. Agr. Food Chem. (1996), 44(6), 1453-1459. The cell walls alreadycontain the polysaccharide complement, and contain their ownperoxidases. Compatible phenolic monomers and a supply of H₂O₂ are theonly requirements to effect in muro lignification. When normalmonolignols are fed, the lignins are structurally extremely similar tothose in the analogous growing plant. A representative protocol is asfollows: Primary cell walls (˜1.2 g dry weight) isolated from 14 d oldmaize cell suspensions were stirred in 120 mL of HOMOPIPES buffer (25mM, pH 5.5 with 4 mM CaCl₂) and artificially lignified over ˜24 h byadding separate solutions of lignin precursors (250 mg in 70 mL of 35%(v/v) dioxane:water) and H₂O₂ (30%, 225 μL in 90 mL water, 1.1 eq) at 3mL/h. In two separate runs of the experiment, guaiacyl-type ligninprecursors consisting of 0, 20, 40, 60% and perhaps 100% (depending onthe monomer), by weight, of monomer-substitute of interest mixed withconiferyl alcohol. Copolymerization of novel monomers with a mixture ofconiferyl and sinapyl alcohols to form mixed syringyl-guaiacyl-basedlignins may also be conducted (to better mimic lignification in dicots).Nonlignified controls are stirred in a solvent mixture similar to thefinal makeup of the lignification reaction media. Cell wall peroxidaseactivity during lignification is monitored with guaiacol-H₂O₂ staining.Although ideal for grasses, this system likely underestimates theimprovements that can be realized in other angiosperms or ingymnosperms; for example, in chemical pulping, grass cell walls arealready significantly more alkali-soluble than in other types of plants.It is therefore also planned to implement and use similarsuspension-cultured systems such as the secondary wall producing systemsfrom tobacco, poplar, as well as Wagner's pine tracheary element system.Such systems provide assorted lignified plant cell walls. See,respectively, Blee, et al. Planta (2001), 212(3), 404-415; Ohlsson, etal. Protoplasma (2006), 228(4), 221-229; and Wagner, et al. Proc. Natl.Acad. Sci. (2007), 104(28), 11856-11861.

Delineating Resultant Lignin Structure:

An important aspect of this work is in establishing how well the novelmonomer incorporated into lignin. With model data from the modelcoupling reactions, NMR methods in particular, and degradative methodssuch as analytical thioacidolysis and the DFRC method, enabledelineating how well incorporated a novel monomer becomes, and into whattypes of structures it is incorporated. See, respectively, Lapierre, etal., Application of new methods for the investigation of ligninstructure. In Forage Cell Wall Structure and Digestibility, Jung, H. G.;Buxton, D. R.; Hatfield, R. D.; Ralph, J., Eds. ASA-CSSA-SSSA: Madison,Wis., (1993), pp 133-166; and Lu, et al., J. Agr. Food Chem. (1997),45(7), 2590-2592. This provides particularly important data fordelineating whether plant alteration has been successful. In addition tocarefully evaluated individual spectra, emerging cell wall 2D NMR“fingerprint” profiles and chemometrics methods can be used to relatethe detailed structural information available in the profile to variousconversion parameters. See Lu & Ralph, Plant J. (2003), 35(4), 535-544;Hedenström, et al. Molecular Plant (2009) 2(5), 933-942.

Testing Biomass Processing:

More straightforward but no less important is the processing and testingof the cell walls with modified lignins to assess the impact of thelignification changes on biomass conversion efficiency. These processesare all well established and won't be detailed herein. For example, theethanolysis process for producing cellulose that is ideal forsaccharification and fermentation. See Pan, X. J. et al. Biotechnol.Bioeng. (2005), 90(4), 473-481.

The walls from above will be subjected to various biomass processingconditions, and compared to controls. To develop a comprehensivedatabase of conditions, at least the following processing pretreatmentsshould be tested: ethanolysis (and other organosolv methods; Pan, X. J.,supra), aminolysis, including the AFEX (ammonia fiber expansion) process(Holtzapple, et al. Appl. Biochem. Biotechnol (1992), 0273-2289),alkaline pulping, and acid hydrolysis. An example protocol forprocessing via alkaline pulping is as follows: The alkaline solubilityof lignins is determined by incubating cell walls under N₂ atmospherewith 100 mL/g of 0.5 M aqueous NaOH for 22 h at 30° C. or for 2.5 h at100 or 160° C. Anthraquinone (0.02 mg/mL) is added to catalyze thehydrolysis of lignin ether inter-unit linkages at 160° C. After cooling,alkaline residues are pelleted (5,000×g, 15 min), resuspended in water,neutralized with acetic acid, and then repeatedly pelleted (5,000×g, 15min) and resuspended in water before freeze-drying and weighing.Original cell walls and alkaline residues are analyzed for lignin by theacetyl bromide method. Alkaline hydrolysates are extracted into ethylacetate containing 50 mM QAM, dried, and dissolved in THF to determinethe molecular weight distribution of by SEC-HPLC. Various GC-MS analysesmay also be performed depending on the monomer used. Enzymaticsaccharification and simultaneous saccharification and fermentation willbe compared on the products from these pretreatments, as well asdirectly on steam exploded material. One of the most convenient andhigh-throughput measures of ruminal fermentability, a gas-accumulationin vitro method, has been shown to correlate well with thefermentability of cellulosic biomass to ethanol (via simultaneoussaccharification and fermentation).

Utility:

As shown in the Examples below, the initial results are quite favorable.For example, the potential energy savings afforded by the remarkableprocessing improvements on cell walls is tremendous. Such gains portendenormous potential for sustainable local (and even small-scale)processing without massive facility costs. A conventional pulp milldigester facility currently costs ˜$1 billion, for example. Decreasingthe need to transport low-density plant materials across largedistances, by processing toward higher-density materials locally, canalso be a huge factor in decreasing the total energy requirements ofprocessing, with consequent major impact on reducing greenhouseemissions, particularly if fossil fuels remain in (partial) use fortransport. These lignin-modified materials appear to be exactly whatthis industry requires. The present method thus has the potential todeliver quantum improvements in biomass processing compared to the moreincremental changes that are envisioned from simply perturbing the knownlignin monomer pathways.

In summary, the present method structurally alters lignin by alteringits monomer complement to allow the biomass polysaccharides to be moreefficiently and sustainably utilized.

Coniferyl Ferulate and Sinapyl Ferulate Incorporation into Lignin:

Recent discoveries highlighting the metabolic malleability of plantlignification indicate that lignin can be engineered to dramaticallydiminish its adverse impact on fiber utilization for nutritional andindustrial purposes. Perturbing single genes in the monolignol pathwaycan lead to dramatic shifts in the proportions of normal monolignols (1,2, 3; FIG. 1) polymerized into lignin or elevated incorporation ofpathway intermediates into the polymer. In normal plants, monolignolsdestined for lignin polymerization can also be extensively acylated withacetate, p-hydroxybenzoate, or p-coumarate (Ralph, J.; Lundquist, K.;Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.; Marita, J. M.; Hatfield, R.D.; Ralph, S. A.; Christensen, J. H.; Boerjan, W. Phytochem. ReV. 2004,3, 29-60).

p-Coumarates acylate the γ-position of phenylpropanoid sidechains ofmainly syringyl units in lignin (Ralph, J.; Hatfield, R. D.; Quideau,S.; Helm, R. F.; Grabber, J. H.; Jung, H.-J. G. J. Am. Chem. Soc. 1994,116, 9448-9456; Grabber, J. H.; Quideau, S.; Ralph, J. Phytochemistry1996, 43, 1189-1194). Structural and enzymatic studies suggest thatsyringyl units are enzymatically pre-acylated with p-coumarate prior totheir incorporation into lignin, see Lu, F.; Ralph, J. 13thInternational Symposium on Wood, Fiber, and Pulping Chemistry, Auckland,New Zealand, May 16-19, 2005, APPITA: Auckland, New Zealand, 2005; pp233-237, implicating sinapyl p-coumarate 5 as the logical precursor.Based on the analysis of isolated lignins and whole cell walls, sinapylp-coumarate could comprise up to 40% of the lignin in some grass tissues(Ralph, J.; Hatfield, R. D.; Quideau, S.; Helm, R. F.; Grabber, J. H.;Jung, H.-J. G. J. Am. Chem. Soc. 1994, 116, 9448-9456; and Hatfield, R.D.; Wilson, J. R.; Mertens, D. R. J. Sci. Food Agric. 1999, 79,891-899). p-Coumarate esters on lignin form few cross-linked structuresmediated by radical coupling reactions and most remain as terminal unitswith an unsaturated side chain and a free phenolic group (Ralph, J.;Hatfield, R. D.; Quideau, S.; Helm, R. F.; Grabber, J. H.; Jung, H.-J.G. J. Am. Chem. Soc. 1994, 116, 9448-9456).

In contrast to p-coumarate, ferulates 4 esterified by simple alcohols,sugars, soluble pectins, or insoluble cell-wall xylans readily undergodiverse radical coupling reactions with each other and with ligninmonomers and oligomers to form cross-linked networks (Oosterveld, A.;Grabber, J. H.; Beldman, G.; Ralph, J.; Voragen, A. G. J. Carbohydr.Res. 1997, 300, 179-181; Grabber, J. H.; Hatfield, R. D.; Ralph, J.;Zon, J.; Amrhein, N. Phytochemistry 1995, 40, 1077-1082; Grabber, J. H.;Ralph, J.; Hatfield, R. D. J. Agric Food. Chem. 2000, 48, 6106-6113;Grabber, J. H.; Ralph, J.; Hatfield, R. D. J. Agric. Food Chem. 2002,50, 6008-6016; and Ralph, J.; Helm, R. F.; Quideau, S.; Hatfield, R. D.J. Chem. Soc., Perkin Trans. 1 1992, 2961-2969). Once polymerized intolignin, ferulate cannot be fully released by solvolytic methods(Grabber, J. H.; Ralph, J.; Hatfield, R. D. J. Agric Food. Chem. 2000,48, 6106-6113). Cleavage of ferulate ester linkages, however,contributes to the unusually high extractability of grass lignin and thedramatically improved enzymatic degradability of grass cell wallsfollowing mild alkaline treatments (Fahey, G. C., Jr.; Bourquin, L. D.;Titgemeyer, E. C.; Atwell, D. G. In Forage Cell Wall Structure andDigestibility; Jung, H. G., Buxton, D. R., Hatfield, R. D., Ralph, J.,Eds.; Am. Soc. Agronomy: Madison, Wis., 1993; pp 715-766).

Ferulate-monolignol ester conjugates, such as coniferyl ferulate 6 orsinapyl ferulate 7 have not been identified in lignins, but they arenaturally produced by some plants as secondary metabolites during, amongother things, lignan biosynthesis (Paula, V. F.; Barbosa, L. C. A.;Howarth, O. W.; Demuner, A. J.; Cass, Q. B.; Vieira, I. J. C.Tetrahedron 1995, 51, 12453-12462; Seca, A. M. L.; Silva, A. M. S.;Silvestre, A. J. D.; Cavaleiro, J. A. S.; Domingues, F. M. J.;Pascoal-Neto, C. Phytochemistry 2001, 56, 759-767; Hsiao, J. J.; Chiang,H. C. Phytochemistry 1995, 39, 899-902; and Li, S. L.; Lin, G.; Tam, Y.K. Planta Med. 2005, 72, 278-280). This raises the exciting possibilitythat plants could be engineered to produce and transport coniferyl orsinapyl ferulate to the apoplastic space in a manner analogous tosinapyl p-coumarate, but with full incorporation of both the ferulateand the monolignol moieties of the conjugate into lignin (FIG. 2).Incorporating these conjugates or other related diphenolics will improvelignin extraction during alkaline pulping (Baucher, M.; Halpin, C.;Petit-Conil, M.; Boerjan, W. Crit. ReV. Biochem. Mol. Biol. 2003, 38,305-350), via one or more of the following mechanisms: (1) cleavage ofester inter-unit linkages to depolymerize lignin; (2) improved ligninsolubility due to ionization of ferulic acid groups; and (3) a smallerinherent size of lignins due to a steady supply of ferulates acting asnew initiation sites for polymerization. Such lignins likely would alsodegrade more readily by acidic or alkaline processes used to pretreatlignocellulosic biomass for saccharification and fermentation to ethanol(Dien, B. S.; Jung, H. J. G.; Vogel, K. P.; Casler, M. D.; Lamb, J. F.S.; Iten, L.; Mitchell, R. B.; Sarath, G. Biomass Bioenergy 2006, 30,880-891; and Murnen, H. K.; Balan, V.; Chundawat, S. P. S.; Bals, B.;Sousa, L. D.; Dale, B. E. Biotechnol. Prog. 2007, 23, 846-850). Becauseof these benefits, a well-developed biomimetic cell-wall model system(Grabber, J. H. Crop Sci. 2005, 45, 820-831) was used to test whetherbioengineering of plants to incorporate coniferyl ferulate into ligninwould enhance the delignification and enzymatic hydrolysis of cellwalls.

EXAMPLES

The following Examples are included solely to provide a more completedisclose of the invention disclosed and claimed herein. The Examples donot limit the scope of the claimed invention in any fashion.

Cell Wall Lignification:

Freshly isolated primary cell walls (˜1.05 g dry weight) from maize cellsuspensions (Grabber, J. H.; Ralph, J.; Hatfield, R. D.; Quideau, S.;Kuster, T.; Pell, A. N. J. Agric. Food Chem. 1996, 44, 1453-1459) werestirred in 120 mL of homopiperazine-N,N′-bis-2-ethanesulfonic acid(Homopipes) buffer (25 mM, pH 5.5 with 4 mM CaCl₂) and artificiallylignified by adding separate solutions of lignin precursors (250 mg in70 mL of 35% (v/v) dioxane/water) and H₂O₂ (30%, 225 μL in 90 mL water,˜1.4 equiv) at 3 mL/h. Precursor mixtures were comprised of coniferylalcohol substituted with 0, 20, 40, or 60% (by weight) ofconiferyl-ferulate. Precursor treatments were replicated by carrying outtwo independent runs of the experiment. Precursors were also prepared indioxane-water to maintain coniferyl ferulate in stable solutions. Ligninprecursors were synthesized as described previously (Lu, F.; Ralph, J.J. Agric. Food Chem. 1998, 46, 2911-2913; and Lu, F.; Ralph, J. J.Agric. Food Chem. 1998, 46, 1794-1796). Nonlignified controls werestirred in a solvent mixture similar to the final makeup of thelignification reaction media. Cell wall peroxidase activity duringlignification was monitored with guaiacol-H₂O₂ staining (Grabber, J. H.;Lu, F. Planta 2007, 226, 741-751). After additions were completed, cellwalls were stirred for an additional 72 h before collection onglass-fiber filters (1.2 μm retention) and washed with water followed byacetone to remove nonincorporated lignin precursors. After evaporatingoff acetone in a hood, cell walls were dried at 55° C. and weighed.Filtrates were evaporated in vacuo to remove acetone and extracted withethyl acetate to isolate nonincorporated precursors (and their couplingproducts). Ethyl acetate extracts were dried with anhydrous magnesiumsulfate, filtered, evaporated in vacuo, and weighed. Nonincorporatedprecursors were then dissolved in DMSO and analyzed by ¹H NMR. Inseparate experiments, dehydrogenation polymers for use as analyticalstandards were formed in high yield (>94%) by slowly adding separatesolutions of the aforesaid series of lignin precursors and H₂O₂ tostirred flasks containing HOMOPIPES buffer and horseradish peroxidase.

Similarly, in parallel runs, primary cell walls (˜1.2 g dry weight)isolated from 14 day-old maize cell suspensions were stirred in 120 mlof Homopipes buffer (25 mM, pH 5.5 with 4 mM CaCI₂) and artificiallylignified over 24 hours by adding separate solutions of ligninprecursors (250 mg in 70 ml of 35% (v/v) dioxane:water) and H₂O₂ (30%,225 μl in 90 ml water) at 3 ml/hour. In two separate runs of theexperiment, guaiacyl-type lignin precursors consisted of 0, 20, 40 or60% (by weight) of coniferyl ferulate mixed with coniferyl alcohol. In athird run, lignin precursors consisted of 0, 20, 40 or 60% (by weight)of sinapyl ferulate mixed with sinapyl and coniferyl alcohols. Thequantities of the monolignols were adjusted to maintain an overallsyringyl to guaiacyl unit ratio of 1:3 in each treatment. Non-lignifiedcontrols were stirred in a solvent mixture similar to the final makeupof the lignification reaction media. Cell wall peroxidase activityduring lignification was monitored with guaiacol-H₂O₂ staining.

The alkaline solubility of lignins was determined by incubating cellwalls under N₂ atmosphere with 100 ml/g of 0.5M aqueous NaOH for 22hours at 30° C. or for 2.5 hours at 100° or 160° C. Anthraquinone (0.02mg/ml) was added to catalyze the hydrolysis of lignin ether inter-unitlinkages at 160° C. After cooling, alkaline residues were pelleted(5,000×g, 15 min), resuspended in water, neutralized with acetic acid,and then repeatedly pelleted (5,000×g, 15 min) and resuspended in waterbefore freeze-drying and weighing. Original cell walls and alkalineresidues were analyzed for lignin by the acetyl bromide method. Alkalinehydrolysates were extracted into ethyl acetate containing 50 mM QAM,dried, and dissolved in tetrahydrofuran (THF) to determine the molecularweight distribution of extracted lignins by SEC-HPLC. After adding2-hydroxycinnamic acid as an internal standard, alkaline hydrolysatesfrom the 30° C. incubations were acidified with HCl, extracted withethyl ether, and silylated for GLC-FID analysis of ferulates anddiferulates.

Cell Wall and Statistical Analyses:

The alkaline solubility of lignins was determined by incubating cellwalls at 30° C. for 24 h, 100° C. for 2.5 h, or 160° C. for 2.5 h insealed Teflon vials under N₂ using 0.5 M aqueous NaOH added at 100 mL/gof cell walls. Anthraquinone (0.02 mg/mL) was added to catalyze thehydrolysis of lignin ether interunit linkages at 160° C. (Kubes, G. J.;B. I., F.; MacLeod, J. M.; Bolker, H. I. Wood Sci. Technol. 1980, 14,207-228). After cooling, alkali-insoluble residues were pelleted(5000×g, 15 min), resuspended in water, neutralized with acetic acid,and then repeatedly pelleted (5000×g, 15 min) and resuspended in waterbefore freeze-drying and weighing. Alkaline hydrolysates recovered fromcell walls (and from dehydrogenation polymers subjected to the sameseries of alkaline treatments) were scanned from 250 to 400 nm with aspectrophotometer. Alkaline hydrolysates were also extracted into ethylacetate containing 50 mM tricaprylylmethyl ammonium chloride, dried, anddissolved in THF to determine the molecular weight distribution ofextracted lignins by size exclusion high-performance liquidchromatography (SECHPLC) (Majcherczyk, A.; Huttermann, A. J.Chromatogr., A 1997, 764, 183-191). After adding 2-hydroxycinnamic acidas an internal standard, alkaline hydrolysates from the 30° C.incubations were acidified with HCl, extracted with ethyl ether, andsilylated for gas-liquid chromatography with flame-ionization detection(GLC-FID) analysis of ferulates and diferulates using previouslydetermined response factors (Grabber, J. H.; Ralph, J.; Hatfield, R. D.J. Agric Food. Chem. 2000, 48, 6106-6113). Response factors offerulate-coniferyl alcohol dimers were assumed to be similar todiferulates.

Cell walls and alkali-insoluble residues were analyzed for lignin by theacetyl bromide method (Hatfield, R. D.; Grabber, J. H.; Ralph, J.; Brei,K. J. Agric. Food Chem. 1999, 47, 628-632), using dehydrogenationpolymers as standards. Cell walls were also analyzed for acid-insolublelignin by the Klason method (Hatfield, R. D.; Jung, H. G.; Ralph, J.;Buxton, D. R.; Weimer, P. J. J. Sci. Food Agric. 1994, 65, 51-58). Wholecell walls (˜40 mg) were sonicated in 1-2 mL of DMSO-d6 and subjected togel-state NMR using a cryoprobe 750 MHz (DMX-750) Bruker Biospin(Rheinstetten, Germany) instrument as described by Kim et al, Bioenerg.Res. 2008, 1, 56-66.

Original cell walls and alkali-insoluble residues collected from 30° C.alkaline incubations were suspended (0.5% w/v) in 20 mM MES buffer (pH5.5, 40° C.) and hydrolyzed with a mixture of “CELLUCLAST 1.5 L” enzyme,“VISCOZYME L” enzyme (each added at 80 μL/g cell wall), and “BIOFEEDBETA” enzyme (added at 80 mg/g cell wall). These are commercial enzymesmarketed by Novo Nordisk, Bagsvaerd Denmark. This mixture of enzymes wasselected to provide a broad complement of cellulase, hemicellulase, andpectinase activities for degrading lignocellulosic material (Grabber, J.H.; Ralph, J.; Hatfield, R. D. J. Agric. Food Chem. 1998, 46,2609-2614). After 2 and 48 h of enzymatic hydrolysis, residues werepelleted by centrifugation (2 min, 10000×g) and an aliquot of thesupernatant was analyzed for uronosyls by a colorimetric method(Blumenkrantz, N.; Asboe-Hansen, G. Anal. Biochem. 1973, 54, 484-489;and Shea, E. M.; Hatfield, R. D. J. Agric. Food Chem. 1993, 41, 380-387)and for neutral sugars by a Dionex BioLC (Dionex Corporation, Sunnyvale,Calif.), (Hatfield, R. D.; Weimer, P. J. J. Sci. Food Agric. 1995, 69,185-196).

Data were analyzed according to a randomized complete block design withtwo replications using PROC GLM (SAS, PC Windows Version 9.1.3; SASInstitute Inc.: Cary, N.C., 2003). Means were subjected to pair-wisecomparisons by the LSD procedure when a significant F-test was detectedat P<0.05. Unless noted otherwise in the text, all reported differenceswere significant at P<0.05.

Results and Discussion of Examples:

Cell Wall Lignification.

In the Examples, varying proportions of coniferyl alcohol and coniferylferulate were polymerized into nonlignified primary walls of maize viawall-bound peroxidase and exogenously supplied H₂O₂. Previous work hasdemonstrated that artificial lignins formed by this system arestructurally similar to those naturally formed in grasses (Grabber, J.H.; Ralph, J.; Hatfield, R. D.; Quideau, S.; Kuster, T.; Pell, A. N. J.Agric. Food Chem. 1996, 44, 1453-1459). Based on mass-balancecalculations, the incorporation of precursors into wall-bound lignindeclined from 92 to 72% in the first and from 100 to 94% in the secondrun of the experiment as the proportion of coniferyl ferulate increasedfrom 0 to 60%. While both runs were conducted in an identical manner,guaiacol staining (data not shown) revealed that poorer incorporation ofprecursors in the first run of the experiment was associated with lowercell wall peroxidase activity at the end of lignification. As noted in aprevious study with a structurally related sinapyl p-coumarate ester(Grabber, J. H.; Lu, F. Planta 2007, 226, 741-751), depressedincorporation of precursors associated with coniferyl ferulate additionwas related to an accelerated loss of cell wall peroxidase activity.Because nonbound apoplastic peroxidases were removed prior to artificiallignification (Grabber, J. H.; Ralph, J.; Hatfield, R. D.; Quideau, S.;Kuster, T.; Pell, A. N. J. Agric. Food Chem. 1996, 44, 1453-1459),peroxidase inactivation would be more markedly manifested in the modelsystem than in plants.

Based on ¹H NMR analysis (data not shown), nonbound precursors recoveredafter lignification were ˜1.4-fold enriched in ferulate compared to theoriginal precursor mixture, indicating that coniferyl ferulate wasincorporated somewhat less efficiently than coniferyl alcohol into wallbound lignins. Extensive copolymerization of coniferyl ferulate intocell wall lignins was, however, readily apparent by gel-state 2D NMR ofwhole cell walls (data not shown) and from UV spectra of alkali-solublelignins fully solubilized at 160° C. from cell walls (FIG. 3).

The average mass-balance lignin content of cell walls declinednumerically from 186 to 164 mg/g as the proportion of coniferyl ferulateincreased (see Table 1 shown in FIG. 4). Due to the incorporation ofmatrix components into lignin (Grabber, J. H.; Ralph, J.; Hatfield, R.D.; Quideau, S.; Kuster, T.; Pell, A. N. J. Agric. Food Chem. 1996, 44,1453-1459), cell walls lignified with coniferyl alcohol had higherKlason and acetyl bromide lignin concentrations than that predicted bymass balance calculations. The Klason and acetyl bromide methods alsoindicated a greater decline in lignin content due to coniferyl ferulateaddition. For the acid-insoluble Klason method, this decline is due toester cleavage and loss of free ferulic acid from lignins formed withconiferyl ferulate. The spectrophotometric acetyl bromide method issensitive to changes in lignin composition, including the presence ofp-hydroxycinnamate esters on lignin (Fukushima, R. S.; Hatfield, R. D.J. Agric. Food Chem. 2004, 52, 3713-3720). To account for shifts in UVabsorption coefficients in the acetyl bromide assay, dehydrogenationpolymers prepared with 0-60% coniferyl ferulate were used as standards.While these polymers provide a good estimate, this may not fully accountfor spectral properties of lignin formed in cell walls.

Ferulate Composition of Cell Walls.

Cell walls were incubated in aqueous NaOH near room temperature for 24 hto cleave and quantify ester-linked p-hydroxycinnamates in cell walls(Hartley, R. D.; Morrison III, W. H. J. Sci. Food Agric. 1991, 55,365-375). Prior to artificial lignification, alkaline hydrolysisreleased 0.4 mg/g of p-coumarate, 8.4 mg/g of ferulate, and 4.7 mg/g ofdiferulates from nonlignified cell walls. Due to their extensivecopolymerization into lignin by alkali-stable bonds (Grabber, J. H.;Ralph, J.; Hatfield, R. D. J. Agric Food. Chem. 2000, 48, 6106-6113),extremely low levels of alkali-labile ferulate and diferulates werereleased from xylans in cell walls lignified with only coniferyl alcohol(Table 1, shown in FIG. 4). Lignifying cell walls with coniferylferulate dramatically increased the amount of ferulate and, to a lesserdegree, diferulate released by alkali. Lignifying cell walls withconiferyl ferulate also considerably increased the quantity ofalkali-labile ferulate cross-coupled to coniferyl alcohol. As notedpreviously with ferulate xylan esters (Grabber, J. H.; Ralph, J.;Hatfield, R. D. J. Agric. Food Chem. 2002, 50, 6008-6016),4-O-cross-coupled dimers predominated over 8-, and 5-dimers (data notshown). Assuming similar GC response factors for dimers, it appears thatcomparable amounts of alkali-releasable ferulate underwent homocouplinginto diferulates vs heterocoupling into cross-coupled ferulate-coniferylalcohol dimmers (Table 1, shown in FIG. 4).

Following lignification with coniferyl alcohol, alkali released 9% ofthe ferulates linked to cell wall xylans as ferulate monomers,diferulates, or cross-product dimers (Table 1, shown in FIG. 4). As thequantity of coniferyl-ferulate increased from 20 to 60% of the precursormixture, the proportion of alkali-labile ferulates derived from cellwall xylans and lignin-incorporated coniferyl ferulate increased from 12to 18%. Fortuitously, the source of these alkali-labile ferulates can beestimated from (Z)-ferulate levels. Nonlignified maize cell wallscontained ˜1.6 mg/g of alkali-labile (Z)-ferulates (as monomers orcoupled as (E,Z)-diferulates) in addition to the predominant(E)-ferulate isomers (data not shown). Because only the (E)-isomer ofconiferyl ferulate was used to lignify cell walls, reductions in thequantity of (Z)-ferulates released by alkali can be used as a generalindicator of ferulate xylan ester incorporation into lignin via alkalistable bonds. In cell walls lignified with coniferyl alcohol, 92% of(Z)-ferulate was incorporated into lignin, which corresponds closely tothe 91% overall incorporation of all ferulate monomers and dimers intolignin. As the proportion of coniferyl ferulate increased from 0 to 60%,the incorporation of (Z)-ferulate into lignin dropped from 92 to 60%. Ifthis decline is typical, then cell wall xylans contributed roughly 45%of the ferulate monomers, diferulates, and cross-product dimers releasedby alkali from cell wall lignified with coniferyl ferulate. Thus, addingconiferyl ferulate with monolignols disrupted ferulate xylan esterincorporation into lignin in a manner analogous to that observed with astructurally related conjugate, sinapyl p-coumarate (Grabber, J. H.; Lu,F. Planta 2007, 226, 741-751). These calculations also indicate thatabout 90% of the ferulate moieties in coniferyl ferulate wereoxidatively coupled to lignin oligomers or polymers. Therefore, ferulatemoieties in coniferyl ferulate readily copolymerized into lignin andtheir addition significantly reduced ferulate xylan cross-linking ofcell walls.

Delignification of Cell Walls.

Various treatments have been developed to delignify herbaceous or woodybiomass for fermentative processes or pulp production (Dien, B. S.;Iten, L. B.; Skory, C. D. Handbook of Industrial Biocatalysis; CRC PressLLC: Boca Raton, Fla., 2005; pp 1-11; Shatalov, A. A.; Pereira, H.Bioresour. Biotechnol. 2005, 96, 865-872; Gratzl, J. S.; and Chen, C. L.Lignin: Historical, Biological, and Materials PerspectiVes; ACSSymposium Series; American Chemical Society: Washington, D.C., 2000;Vol. 742, pp 392-421). Because lignins containing ester inter-unitlinkages should be readily cleaved by alkali, 0.5 M aqueous NaOH wasused to study how the incorporation of coniferyl ferulate affectscell-wall delignification. A 30° C. treatment for 24 h was chosen torepresent a mild alkaline pretreatment of biomass for ethanolfermentation (Dien, B. S.; Iten, L. B.; Skory, C. D. Handbook ofIndustrial Biocatalysis; CRC Press LLC: Boca Raton, Fla., 2005; pp1-11). To represent harsher biomass pretreatments and pulpingconditions, (Dien, B. S.; Iten, L. B.; Skory, C. D. Handbook ofIndustrial Biocatalysis; CRC Press LLC: Boca Raton, Fla., 2005; pp 1-11;Shatalov, A. A.; Pereira, H. Bioresour. Biotechnol. 2005, 96, 865-872;and Gratzl, J. S.; Chen, C. L. Lignin: Historical, Biological, andMaterials PerspectiVes; ACS Symposium Series; American Chemical Society:Washington, D.C., 2000; Vol. 742, pp 392-421) refluxing at 100° C. andcooking at 160° C. for 2.5 h was used. Refluxing at 100° C. generallycleaves recalcitrant ester inter-unit linkages and fully solubilizesnonbound lignins, while cooking at 160° C. for 2.5 h with anthraquinoneshould solubilize additional lignin by cleaving ether inter-unitlinkages. In these studies, NaOH concentrations, temperature, time, andanthraquinone levels were not optimized or meant to fully mimicpotential commercial practices. The conditions were selected merely toillustrate how coniferyl ferulate incorporation into lignin affects theease of cell-wall delignification by alkali.

The proportion of lignin solubilized with aqueous 0.5 M NaOH increasedwith the severity of hydrolysis conditions and with the proportion ofconiferyl ferulate used to form lignin (see FIGS. 5A and 5B). At 30° C.,alkali-soluble lignin, as a proportion of cell-wall lignin increasedquadratically from 32 to 66% as coniferyl ferulate increased from 0 to60% of precursors. At higher temperatures, coniferyl ferulate additionslinearly increased alkali-soluble lignin from 57 to 93% at 100° C., andfrom 69 to 99% at 160° C. While alkaline extraction of about 70% oflignin required heating at 160° C. for cell walls lignified withconiferyl alcohol, heating at 100° C. sufficed if coniferyl ferulatecomprised about 30% of lignin precursors. Alternatively, coniferylferulate addition also permitted more extensive delignification of cellwalls at normal 160° C. cooking temperatures. Because coniferyl ferulatereduced the amount of lignin formed in cell walls it had less effect onthe total quantity of alkali-soluble lignin released particularly at160° C. (see Table 2, shown in FIG. 6).

Incorporation of coniferyl ferulate into lignins dramatically improvedthe delignifcation of walls under both mild and severe pulpingconditions (FIGS. 5A and 5B). Typical pulping conditions (160° C. withanthraquinone for 1.5 h) removed 70% of the lignin from “normal” wallslignified with coniferyl alcohol. The data in FIG. 5A indicate a similardegree of delignification can be obtained under refluxing conditions(100° C. for 2.5 h) if lignins are formed with ˜25% coniferyl ferulate.A similar degree of delignification may be obtained under even milderconditions (30° C. for 20 h) if lignins are formed with 60% coniferylferulate (see dotted line and arrows in FIG. 5A). Up to 40% of thelignins in grasses are formed by a related conjugate (sinapylp-coumarate), so similar levels of coniferyl ferulate incorporation areplausible. The arrows in FIG. 5B indicate the fiber yields at 70%delignification. Higher pulp yields can be anticipated because of theeasier delignification. These results show that incorporating coniferylferulate into the lignin allows the lignin to be disassembled using muchgentler conditions.

Increased severity of delignification and greater proportions ofconiferyl ferulate reduced the molecular weight of lignin released fromcell walls. As the severity of delignification conditions increased, theproportion of oligomeric (2.5-17.5 kDa) and polymeric (>17.5 kDa)lignins declined, while the proportion of trimers and smaller fragments(<0.6 kDa) increased (FIG. 7). For all delignification treatments,incorporation of coniferyl ferulate into lignin mainly increased theproportion monomers and dimers (<0.4 kDa) and decreased the proportionof oligomeric (2.5-17.5 kDa) and polymeric (>17.5 kDa) lignins releasedfrom cell walls (FIG. 8). Consequently, incorporation of an esterinterunit linkage into lignin via coniferyl ferulate enhanced alkalinedepolymerization of lignin, leading to a greater release of lignin fromcell walls. Conversely, ferulate's ability to act as an initiation sitefor lignin polymerization (Grabber, J. H.; Ralph, J.; Hatfield, R. D. J.Agric. Food Chem. 2002, 50, 6008-6016; Ralph, J.; Grabber, J. H.;Hatfield, R. D. Carbohydr. Res. 1995, 275, 167-178) could mean thatcontinual coniferyl ferulate addition truncated polymerization to yieldmore numerous and smaller lignin chains than would be obtained withnormal monolignols. In either case, cleavage of ester inter-unitlinkages or a lower inherent size of polymers both contribute toenhanced solubilization of cell wall lignins formed with coniferylferulate.

Due to greater lignin extractability and lower intrinsic lignin levels,the lignin content of alkali-insoluble residue (AIR) at each temperaturedropped dramatically as the proportion of coniferyl ferulate increased(FIG. 9). Thus, while a 160° C. alkaline hydrolysis of walls lignifiedwith coniferyl alcohol yielded AIR with 123 mg/g of lignin, AIR withcomparable lignin levels could be obtained at 100° C. with ˜30%coniferyl ferulate. Alternatively, heating cell walls lignified with˜30% of coniferyl ferulate at 160° C. yields AIR with much lower ligninconcentrations. As a result, incorporation of coniferyl ferulate intolignin enables pulping at lower temperatures or pulping at hightemperature with reduced cooking time, and likely eliminating the needfor bleaching.

Yields of alkali-soluble carbohydrate (ASC) increased and AIR decreased,as coniferyl ferulate comprised a greater proportion of lignin. (Table2, shown in FIG. 6). The response of these fractions tended to be mostpronounced at low to moderate levels of coniferyl ferulate addition andat higher hydrolysis temperatures. As hydrolysis temperatures increasedto 160° C., the recovery of AIR from nonlignified cell walls and cellwalls lignified with 40% of coniferyl ferulate leveled off near thecellulose content of cell walls (˜250 mg/g) (Grabber, J. H.; Ralph, J.;Hatfield, R. D. J. Agric. Food Chem. 1998, 46, 2609-2614). Becauseconiferyl ferulate renders lignin more extractable by alkali, it wouldfollow that pectin and hemicellulose extraction would improve as well.Indeed, lignin-degrading pretreatments are often used to improve theextractability of noncellulosic polysaccharides for analysis(Selvendran, R. R.; Stevens, B. J. H.; O'Neill, M. A. In Biochemistry ofPlant Cell Walls; Brett, C. T., Hillman, J. R., Eds.; CambridgeUniversity Press: Cambridge, 1985; pp 39-78.(41) Grabber, J. H.;Hatfield, R. D.; Ralph, J. J. Agric. Food Chem. 2003, 51, 4984-4989).Hence, at a given temperature, delignification of cell walls containingconiferyl ferulate yields AIR with less noncellulosic and, as mentionedabove, less lignin contamination. Alternatively, coniferyl ferulateprovides the option of delignifying cell walls under milder conditionsto increase total fiber yields. For example, cell walls lignified withconiferyl alcohol yielded 497 mg/g of AIR at 160° C. compared to 749mg/g of AIR at 30° C. for cell wall lignified with 40% coniferylferulate (Table 2, FIG. 6); both types of AIR contained similar amountsof lignin (˜130 mg/g, FIG. 9).

Enzymatic Hydrolysis of Cell Walls and Alkali Insoluble Residues.

Cell walls and alkali-insoluble residues were incubated with highloadings of fibrolytic enzymes to assess whether coniferyl ferulateincorporation into lignin enhances the rate, and above all, the extentof structural polysaccharide hydrolysis. The release of all sugars(i.e., glucose, arabinose, xylose, galactose, uronosyls) respondedsimilarly to coniferyl ferulate incorporation into lignin; therefore,only total carbohydrate yields are reported and discussed below.Incorporation of coniferyl ferulate into lignin improved carbohydrateyields from both cell walls and AIR recovered following treatment withaqueous NaOH at 30° C. (Table 3, shown in FIG. 10). Prior to alkalinepretreatment, yields of carbohydrates from artificially lignified andnonlignified cell walls linearly increased as lignin content declinedafter both 2 and 48 h of enzymatic hydrolysis (FIGS. 11A and 11B,respectively). Thus, coniferyl ferulate enhanced carbohydrate yieldsprimarily by reducing the lignin content of cell walls. Even so, reducedcross-linking of lignin to feruloylated xylans with coniferyl ferulateadditions could also play a role in enhancing cell wall hydrolysis(Grabber, J. H.; Ralph, J.; Hatfield, R. D. J. Agric. Food Chem. 1998,46, 2609-2614).

Pretreatment with NaOH dramatically improved the enzymatic hydrolysis ofcarbohydrates from AIR derived from all types of lignified cell walls(Table 3, shown in FIG. 10). The dramatic degradability response ofgrass cell walls to alkaline pretreatments has been mainly attributed tocleavage of cross-links between lignin and feruloylated xylans and tolignin extraction (Fahey, G. C., Jr.; Bourquin, L. D.; Titgemeyer, E.C.; Atwell, D. G. In Forage Cell Wall Structure and Digestibility; Jung,H. G., Buxton, D. R., Hatfield, R. D., Ralph, J., Eds.; Am. Soc.Agronomy: Madison, Wis., 1993; pp 715-766). Coniferyl ferulate improvedlignin extractability and carbohydrate yields from AIR, but yieldsquickly plateaued even as lignin levels continued to decline (see FIGS.11A and 11B). Indeed, after both 2 and 48 h of enzymatic hydrolysis,maximal carbohydrate yields from lignified AIR were >100 mg/g lower thannonlignified AIR, indicating lignin content per se was not the onlyfactor limiting cell wall hydrolysis.

On a cell wall basis, hydrolytic enzymes released a fairly constantproportion of carbohydrate from AIR derived from nonlignified andlignified cell walls (Table 3, shown in FIG. 10). If ASC are included,then total fermentable carbohydrate yields following alkalinepretreatment were greatest from nonlignified cell walls, intermediatefrom cell walls lignified with coniferyl ferulate, and lowest from cellwalls lignified with coniferyl alcohol. Thus, the benefits of alkalinepretreatment and incorporation of alkali-labile coniferyl ferulate intograss lignins will only be fully realized if noncellulosic ASC arerecovered and utilized for fermentation. This also indicates shifts inlignin alkaline solubility mainly alters the proportion of nondegradablecarbohydrate versus ASC in cell walls without markedly changing the sizeof the degradable AIR fraction in cell walls. While not examined here,coniferyl ferulate incorporation into lignin could also reduce biomassconversion costs if lower enzyme loadings could be used forsaccharification.

Based on these Examples, incorporation of coniferyl ferulate intograminaceous feedstocks reduces lignifications and permits moreefficient delignification and enzymatic hydrolysis of cell walls. Thisin turn reduces inputs for energy, pressure vessel construction, andbleaching during papermaking, and lessens pretreatment and enzyme costsassociated with biomass conversion. Comparable or greater benefits areanticipated for hardwoods, softwoods, and herbaceous dicots that havelower inherent lignin extractability.

In preliminary studies, adding sinapyl ferulate with coniferyl andsinapyl alcohols had comparable effects on lignin formation, ligninextractability, and cell wall degradability. Therefore, geneticengineering of plants to incorporate coniferyl ferulate into guaiacyllignins in softwoods or coniferyl and sinapyl ferulates into mixedsyringyl-guaiacyl-type lignins in hardwoods and herbaceous plants willgreatly enhance the utilization of plant cell walls.

REFERENCES

The following references are incorporated herein by reference:

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What is claimed is:
 1. An isolated lignified plant cell wall comprisingan isolated plant cell wall that includes lignin, wherein the ligninincludes a ferulate residue incorporated therein, wherein the ferulateresidue is a ferulate-monolignol ester conjugate, wherein theferulate-monolignol ester conjugate comprises a monolignol moietyesterified to a ferulate moiety via a γ-carbon on the monolignol moiety,wherein both the monolignol moiety and the ferulate moiety areindependently incorporated within the lignin.
 2. The isolated lignifiedplant cell wall of claim 1, wherein the ferulate residue is selectedfrom a monomer conjugate group consisting of coniferyl ferulate andsinapyl ferulate.
 3. The isolated lignified plant cell wall of claim 1,derived from maize.
 4. The isolated lignified plant cell wall of claim1, derived from a tree.
 5. The isolated lignified plant cell wall ofclaim 4, derived from a tree of the family Myrtaceae, Salicaceae, or ahybrid thereof.
 6. The isolated lignified plant cell wall of claim 5,derived from a tree of a genus selected from the group consisting ofSalix, Eucalyptus, Corymbia, and Angophora, or a hybrid thereof.
 7. Theisolated lignified plant cell wall of claim 1, derived from a eucalyptustree, a poplar tree, a willow tree, or a hybrid thereof.
 8. The isolatedlignified plant cell wall of any one of claims 1-7, wherein the ferulateresidue is derived from coniferyl ferulate.
 9. The isolated lignifiedplant cell wall of any one of claims 1-7, wherein the ferulate residueis derived from sinapyl ferulate.
 10. A method of manufacturing modifiedlignin, the method comprising: conducting a lignin-producingpolymerization reaction in a cell wall in the presence of at least onepolymerizable monomer comprising a ferulate-monolignol ester conjugate,wherein the ferulate-monolignol ester conjugate comprises a monolignolmoiety esterified to a ferulate moiety via a γ-carbon on the monolignolmoiety, wherein at least one of the polymerizable monomers isincorporated into the resulting lignin wherein both the monolignolmoiety and the ferulate moiety are independently incorporated within thelignin.
 11. The method of claim 10, wherein the polymerizable monomer isselected from the group consisting of coniferyl ferulate and sinapylferulate.
 12. The method of claim 10 or claim 11, wherein from about 10%by wt to about 60% by wt of the polymerizable monomers are reacted inthe polymerization reaction.
 13. The method of claim 10 or claim 11,wherein the polymerization reaction is conducted in vitro.
 14. Themethod of claim 13, wherein the polymerization reaction comprisesisolating a cell wall from a plant cell suspension and lignifying thecell wall in the presence of the polymerizable monomer.
 15. The methodof claim 14, wherein the plant cell suspension is isolated from maize.16. The method of claim 14, wherein the plant cell suspension isisolated from a tree of the family Myrtaceae, Salicaceae, or a hybridthereof.
 17. The method of claim 14, wherein the plant cell suspensionis isolated from a tree of a genus selected from the group consisting ofEucalyptus, Corymbia, and Angophora, or a hybrid thereof.
 18. The methodof claim 14, wherein the plant cell suspension is isolated from aeucalyptus tree, a poplar tree, a willow tree, or a hybrid thereof. 19.The method of claim 10 or claim 11, wherein the polymerization reactionis conducted in vivo.
 20. A modified lignin produced by a methodcomprising: conducting a lignin-producing polymerization reaction in acell wall in the presence of at least one polymerizable monomercomprising a ferulate-monolignol ester conjugate, wherein theferulate-monolignol ester conjugate comprises a monolignol moietyesterified to a ferulate moiety via a γ-carbon on the monolignol moiety,wherein at least one of the polymerizable monomers is incorporated intothe resulting lignin wherein both the monolignol moiety and the ferulatemoiety are independently incorporated within the lignin.
 21. Themodified lignin of claim 20 wherein the polymerizable monomer isselected from the group consisting of coniferyl ferulate and sinapylferulate.
 22. The method of claim 20, wherein the polymerizationreaction is conducted in vivo.
 23. The method of claim 20, wherein thepolymerization reaction is conducted in vitro.
 24. The method of claim23, wherein the polymerization reaction comprises isolating a cell wallfrom a plant cell suspension and lignifying the cell wall in thepresence of the polymerizable monomer.